Protein tyrosine phosphatases (PTPs) are important for the regulation of signaling pathways, acting as a biochemical counterbalance to kinases.(1, 2) This mechanism plays diverse roles in biological systems, including the regulation of T cell antigen recognition and activation.(3) As a result, PTPs are an important target for both medicinal chemistry and biochemical research.(4, 5) However, few general strategies have been elucidated for the determination of PTP specificity or inhibitor design.
A well-studied immune cell PTP is CD45. CD45 is a receptor-like PTP (RPTP), and the most prevalent membrane-associated PTP in T cells.(6) Misregulation of CD45 results in severe combined immunodeficiency (SCID), and the receptor is implicated in autoimmune disease. Currently, the primary strategies to examine CD45 activity rely on the use of phosphotyrosine-specific antibodies, previously validated synthetic inhibitors, or the synthesis of phosphopeptide substrates.(7) These strategies severely limit the type and amount of data that can be collected on PTP's since they either are not specific to a particular PTP, as in the case of phosphotyrosine-specific antibodies, or else they require the identification and validation of a new compound, as in the case of synthetic inhibitors. Specific phosphopeptides have been used to study PTP activity, however these compounds require a separate detection strategy, such as an enzyme-linked method.(8) These strategies all suffer from difficulties due to poor signal-to-noise ratios, making it difficult to distinguish positive hits or small changes to PTPs activity. None of these strategies allow for detection by covalent labeling of the active PTPs. Moreover, the study and detection of PTPs is limited because of the lack of known specific inhibitors for desired PTPs having high potency.
The design of specific PTP inhibitors remains a challenge, and new strategies that provide enhanced activity or reduce development time are of continuing interest.(4) Currently, reversible and irreversible inhibitors of PTPs are known.
Many different reversible inhibitors of PTPs have been reported. A classic strategy for designing reversible, competitive PTP inhibitors has exploited non-hydrolyzable phosphotyrosine (pTyr) mimics, such as phosphonomethylphenylalanine (Pmp).(9) It has been previously shown that modification of Pmp to phosphonodifluoromethylphenylalanine (F2Pmp) improves the potency of these derivatives (FIG. 1).(10) Reported derivatives of Pmp include fluoro, difluoro, chloro, and dichloro derivatives.(10-14) These strategies have been successfully applied to develop many different competitive inhibitors for a variety of PTPs.(15) However, these types of PTP inhibitors have many different disadvantages. For example, reversible inhibitors often have low specificity for the target PTP, and can inhibit undesired targets. Many also have low potency (affinity), therefore reducing their utility in medicinal chemistry or biological applications. Reversible PTP inhibitors have limited use in the detection of PTPs in microscopy, histology, proteomic, or diagnostic tests and they cannot be used in enzyme labeling strategies. These disadvantages seriously hinder the study of PTPs in medicinal chemistry and biochemical research.
In recent years, there has been renewed interest in identifying irreversible or covalent inhibitors of a variety of enzymes, including PTPs. In addition to improved potency, irreversible inhibitors (sometimes referred to as suicide substrates) can be of interest in the development of enzyme labeling strategies. For example, irreversible inhibitors, when attached to fluorophores or affinity tags, have been employed as activity-based protein probes (ABPP).(16, 17) Known irreversible inhibitors of PTPs include quinone methides,(18) aryl vinyl sulfonates,(19) nitrostyrene,(20) and α-bromobenzylphosphonate (BBP) derivatives.(21, 22) Other notable strategies have included the synthesis of fluorogenic substrates of PTPs, which should allow improved assay, detection, and imaging applications.(23) Therefore, irreversible PTP inhibitors can provide a means to label and detect enzyme activity with great sensitivity.
Widlanski and coworkers first demonstrated that BBP derivatives could act as irreversible inhibitors of PTPs.(21) Kumar et al. subsequently tested the activity of α-bromobenzylphosphonate (BBP) analogs containing affinity tags to be used as a detection strategy for PTPs using biotin-labeled derivative 2 (FIG. 1).(22) These derivatives were found to form covalent adducts with PTPs, forming the basis of proteomic strategies for PTP identification. However, compound 2 was also shown to covalently label a wide variety of PTPs, establishing a major barrier to its use as a specific labeling agent for PTPs and limiting its application as an inhibitor of specific enzymes. Moreover, the synthesis of compound 2 is difficult, and not easily scalable. Due to its lack of specificity and difficulty in its synthetic preparation, compound 2 does not lend itself to identification of new and specific PTP inhibitors. For example, the compound could not be easily inserted into peptides via peptide synthesis techniques such as solid-phase peptide synthesis (SPPS). It can therefore not be used in the preparation of peptide libraries that can be used to find inhibitors for various PTPs.
Consequently, there is a need for methods and systems which can provide for the synthesis and identification new irreversible inhibitors of PTPs, which are both specific and potent, while avoiding some of the problems listed above. Such methods and systems could be used to expand our knowledge of PTPs, and allow for new methods to detect PTP activity and identify new PTP-specific substrate sequences.